Microchemical Analysis of Nervous Tissue

Methods in Life Sciences

Neville N. Osborne

• Publisher: Pergamon
• Year: 2013
• ISBN:
  9781483187389
• Topic: Biological Sciences
• Subtopic: Human Anatomy & Physiology
• Index: Biological Sciences

Microchemical Analysis of Nervous Tissue focuses on the use of microbiochemical methods in the analysis of nervous tissue, with emphasis on those related to the study of amines, amino acids, phospholipids, and proteins. Special attention is paid to the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. Comprised of 10 chapters, this volume begins with an overview of microprocedures used in neurochemistry, followed by a discussion on the importance of choosing the biological material for microanalysis. The isolation of nervous tissue for analysis is then considered, with particular reference to invertebrate neurons; cell components from fresh, "fixed, " freeze-dried, and frozen impregnated tissue; and discrete areas of nervous tissue. Subsequent chapters describe some instruments and glassware used in microprocedures, along with the applications of such procedures; general techniques used in microprocedures; microdetermination of phospholipids as well as amines and amino acids as dansyl derivatives; and microelectrophoresis of proteins. This book will be of interest to molecular biologists, microbiologists, physiologists, and neurochemists.

CHAPTER 1

General Introduction

Publisher Summary

The physiology, morphology, functional role, and biochemistry of individual neurons are studied and the neurons in the nervous system are related to one another before a real insight is gained into the intricate mechanism of the nervous system. However, progress has been slow basically for two main reasons. First, the majority of neurons are difficult to characterize and study as entities because of their small size, and, second, there is a lack of suitable microprocedures that would permit the study of different biochemical parameters in individual neurons. This chapter describes some microprocedures recently developed in the laboratory. It focuses on the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. The microbiochemical methods are described related especially to the study of amines, amino acids, phospholipids, and proteins. The chapter also describes sensitive microprocedures and their applications are explained for studying small amounts of tissue, for example, isolated cells, discrete areas of brain, and biopsy material.

WHY a monograph on microprocedures in neurochemistry? This is not difficult to justify when one considers that the human brain has approximately 10¹⁰ nerve cells, while the tiny brain of the ant (Formica lugubris) has about 100,000. These vast populations of neurons present a formidable challenge to the biologist trying to understand how the nervous system works. From the mass of electrophysiological and electron microscopical data which has accumulated it can now generally be concluded that nerve cells are independent units (see e.g. Bullock, 1967; Bullock and Horridge, 1965; Eccles, 1964; Segundo, 1970; Horridge, 1968). Furthermore, each neuron has several parts: (1) receptive loci specialized in transducing the dozens of imputs which impinge on them in several ways; (2) pacemaker loci which inject spontaneous rhythms; (3) mixing and integrating loci; (4) threshold loci for initiating all-or-none nerve impulses in bursts and trains from 1 to 1000 per second; and (5) transmitter loci at each of the far ends of the nerve cell, where they influence up to several dozen others. Clearly, biochemical information to be gained from classical studies using relatively large amounts of nervous tissue (and therefore large numbers of cells which may have very different properties) is of limited value. This problem is complicated by the existence of vast numbers of glial cells which form a close and integrated association with the neurons. Obviously the physiology, morphology, functional role and biochemistry of individual neurons have to be studied and the neurons in the nervous system related to one another before a real insight is gained into the intricate mechanism of the nervous system. However, progress has been slow, basically for two main reasons. Firstly, the majority of neurons are difficult to characterise and study as entities because of their small size, and, secondly, there is a lack of suitable microprocedures which would permit the study of different biochemical parameters in individual neurons.

One way of circumventing these difficulties is either to separate disaggregated nervous tissue, thus obtaining populations of neurons and glial (Rose, 1968), or to fractionate homogenates of nervous tissue and secure relatively pure fractions of a constituent part of the different neurons, e.g. the nerve endings (Whittaker, 1973). Studies of this kind have many advantages, but they, too, suffer from certain drawbacks, such as the possibility that changes could occur in the constituents, caused by the elaborate separation or fractionation procedures employed; moreover, any differences there may be in the properties of similar structures obtained from the brain cannot be observed. Another approach is to analyse small defined areas of the nervous system or individual neurons where possible. This presupposes the presence of suitable microchemical procedures. Perhaps a distinction should be made here between macro- (normal), micro- and ultra-procedures, though one might think that such a distinction is meaningless, since they represent a scale continuum.

In theory they do; however, in practice there is a change from macroscale (i.e. brain homogenates) to micro (i.e. one very large neuron, or microquantities of nervous tissue), and over this range many macroprocedures can be modified and scaled down. The next step, the ultra-microprocedure (parts of a single (20 μ) minute nerve cell), is a ‘quantum jump’ and often requires elaborate apparatus and new approaches.

The purpose of this monograph is to describe some microprocedures recently developed in this laboratory. Special attention will be paid to the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. The microbiochemical methods described will be those related especially to the study of amines, amino acids, phospholipids and proteins. Many other extremely sensitive microprocedures (plus-ultra-microprocedures) have been developed within the last thirty years (see Chap. 5) and though their description is beyond the scope of the monograph, a brief review of some of these methods and their applications is presented. Perhaps it should be pointed out that emphasis is often laid only on the applicability of microprocedures for studying small amounts of tissue, e.g. isolated cells, discrete areas of brain, biopsy material, etc., whereas they also have other important virtues. Some micromethods, for example, are less time-consuming than normal procedures, and are for this reason employed even when the material available is unlimited. Moreover, the cost of analysing material by micromethods can often be very much less than that of similar normal scale studies.

References

BULLOCK, T. H. Signals and neuronal coding. In: QUARTON G.C., MELNECHUK T., SCHMITT F.O., eds. The Neurosciences: a Study Program. New York: The Rockefeller University Press; 1967:347–452.

BULLOCK, T. H., HORRIDGE, G. A.Structure and Function in the Nervous Systems of Invertebrates. San Francisco: W. H. Freeman, 1965.

ECCLES, J. C.The Physiology of Synapses. New York: Academic Press, 1964.

HORRIDGE, G. A.Interneurons. San Francisco: W. H. Freeman, 1968.

ROSE, S. P. R. (1968) The biochemistry of neurones and glia. In: Applied Neurochemistry (Eds. A. N. Davison and J. Dobbing), pp. 332–355.

SEGUNDO, J. P. Functional possibilities of nerve cells for communication and for coding. Acta Neurol. Latinoamer. 1970; 14:340–344.

WHITTAKER, V. P. The biochemistry of synaptic transmission. Naturwissenschaften. 1973; 60:281–289.

CHAPTER 2

Choice of Biological Material for Microanalysis

Publisher Summary

This chapter discusses the fact that the mammalian brain contains a great number of neurons, which presents a problem. The difficulty lies in the choice of appropriate experimental objects. In this respect, certain invertebrate nervous systems offer a number of advantages, in that they are organized in an orderly manner, have fewer nerve cells than the vertebrates, have specialized giant neurons, and can be individually characterized. There are certain vertebrate preparations, which contain populations of giant neurons, though they are difficult to characterize individually. Another important advantage of the invertebrate neurons is that they can retain their functional activity after dissection and survive for several hours or even days. This makes it possible to perform in vitro experiments on invertebrate nervous systems, monitoring the activity of individual neurons by means of intra- or extracellular recording while the environment of the cell can be controlled or changed by adding or substituting ions and inhibitors. The chapter also explains that there is not only an enormous variety of invertebrate cell preparations, but also of invertebrate preparations of giant synapses and giant axons, which are suitable for biochemical analysis.

AS previously mentioned, the fact that the mamma...